The objective of the research proposed is to improve our understanding of the mechanisms involved in the biochemistry and regulation of steroid hormone biosynthesis. The proposed studies focus on the cholesterol side chain cleavage reaction, the initial and rate-controlling step in the pathway. Recombinant DNA methods will be used to express human ferredoxin reductase and human cytochrome P450scc in E. coli. The mature forms of the enzymes produced in E. coli will be purified and their properties compared with those of the naturally occurring enzymes. Recombinant human ferredoxin has previously been expressed in E. coli, and this will provide all three enzymes needed for study of the cholesterol side chain cleavage reaction. Site-directed mutagenesis is proposed to produce modified forms of each protein for structural and functional studies. For both ferredoxin reductase and P450scc, amino acid replacements will be made to investigate the role of specific residues in binding and electron transfer interactions with human ferredoxin. For ferredoxin reductase, substitutions will be made to identify residues involved in binding the cofactor, FAD, and the substrate, NADPH. For P450scc, substitutions will be made to identify residues important in determining steroid binding selectivity and specificity of the sites of hydroxylation. In addition, efforts will be made to obtain crystals of the recombinant proteins suitable for X-ray crystallography. The ability to express steroid hydroxylase components in bacteria will make larger amounts of these proteins available for future studies. It will also serve to avoid reliance on human (or other animal) tissues as a source of these proteins and reduce associated costs and risk factors.